Article

CD40 promotes MHC class II expression on adipose tissue macrophages and regulates adipose tissue CD4+ T cells with obesity † † David L. Morris,*,1 Kelsie E. Oatmen, Taleen A. Mergian, Kae Won Cho,*,2 ‡ { Jennifer L. DelProposto,* Kanakadurga Singer,* Carmella Evans-Molina, Robert W. O’Rourke,§, and Carey N. Lumeng*,3 *Department of Pediatrics and Communicable Diseases, University of Michigan Health System, Ann Arbor, Michigan, USA; † ‡ Literature, Science and Arts Program, University of Michigan, Ann Arbor, Michigan, USA; Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana, USA; §Department of Surgery, University of Michigan Medical School, Ann { Arbor, Michigan, USA; Department of Surgery, Ann Arbor Veteran’s Administration Hospital, Ann Arbor, Michigan, USA RECEIVED JANUARY 7, 2015; REVISED OCTOBER 30, 2015; ACCEPTED NOVEMBER 4, 2015. DOI: 10.1189/jlb.3A0115-009R

ABSTRACT CD80 and CD86 expression in obese patients with type Obesity activates both innate and adaptive immune re- 2 diabetes. These findings indicate that CD40 signaling in sponses in adipose tissue, but the mechanisms critical for adipose tissue macrophages regulates major histocom- patibility complex class II and CD86 expression to control regulating these responses remain unknown. CD40/CD40L + signaling provides bidirectional costimulatory signals the expansion of CD4 T cells; however, this is largely between -presenting cells and CD4+ T cells, and dispensable for the development of obesity-induced in- CD40L expression is increased in obese humans. flammation and insulin resistance in mice. J. Leukoc. Biol. 99: 1107–1119; 2016. Therefore, we examined the contribution of CD40 to the progression of obesity-induced in mice. CD40 was highly expressed on adipose tissue macro- phages in mice, and CD40/CD40L signaling promoted Introduction the expression of antigen-presenting cell markers in Chronic adipose tissue inflammation has long been implicated adipose tissue macrophages. When fed a high fat diet, in obesity-associated diseases, such as cardiovascular disease, Cd40-deficient mice had reduced accumulation of con- type 2 diabetes, and metabolic syndrome. Adipose tissue + + + 2 ventional CD4 T cells (Tconv: CD3 CD4 Foxp3 ) in vis- inflammation involves activation of both innate and adaptive ceral fat compared with wild-type mice. By contrast, – + + + immune responses [1, 2]. MHC class II restricted signals from the number of regulatory CD4 Tcells(Treg:CD3CD4 Foxp3+) in lean and obese fat was similar between wild- macrophages and dendritic cells contribute to activation in type and knockout mice. Adipose tissue macrophage response to obesogenic cues and contribute to metabolic – content and inflammatory gene expression in fat did not dysfunction [3 5]. ATMs and dendritic cells can function as differ between obese wild-type and knockout mice; APCs and provide critical cues to generate proinflammatory however, major complex class II and signals that include activation of adipose tissue CD4+ T cells CD86 expression on adipose tissue macrophages was toward a Th1 and effector/memory phenotype [6–8]. Obesity is reduced in visceral fat from knockout mice. Similar results also associated with the induction of CD8+ T cells in adipose were observed in chimeric mice with hematopoietic fl tissue and a decrease in Tregs that contribute to a proin amma- Cd40-deficiency. Nonetheless, neither whole body nor tory environment [9, 10]. Based on this, there has been hematopoietic disruption of CD40 ameliorated obesity- increasing interest in understanding how APC–T cell cross talk induced insulin resistance in mice. In human adipose contributes to activation of an adaptive and tissue, CD40 expression was positively correlated with adipose tissue inflammation in obesity. Costimulatory signals from APCs are important for sustaining Abbreviations: ATM = adipose tissue macrophage, ATT = adipose tissue T cell activation, and recent studies have aimed to elucidate the T cell, BMT = bone marrow transplant, eWAT = epididymal white adipose tissue, roles of costimulatory molecules in adipose tissue inflammation GTT = glucose tolerance test, HFD = high fat diet, HOMA-IR = homeostatic model assessment of insulin resistance, iWAT = inguinal white adipose tissue, ITT = insulin tolerance test, KO = Cd40 knockout, MFI = median fluorescent 1. Current affiliation: Indiana University School of Medicine, Indianapolis, intensity, MHC II = major histocompatibility complex, class II, ND = normal diet, Indiana, USA.

SVC = stromal vascular cell, Tconv = conventional T cell, Treg = regulatory T cell, 2. Current affiliation: Soonchunhyang University, Asan, South Korea. WT = wild-type 3. Correspondence: Department of Pediatrics and Communicable Diseases, University of Michigan Health System, 2057 Biomedical Sciences Research The online version of this paper, found at www.jleukbio.org, includes Building, 109 Zina Pitcher Place, Ann Arbor, MI 48109-2200, USA. E-mail: supplemental information. [email protected]

0741-5400/16/0099-1107 © Society for Leukocyte Biology Volume 99, June 2016 Journal of Leukocyte Biology 1107 because these may be novel therapeutic targets for mitigating dietary obesity. These findings indicate that CD40 signaling obesity-induced metabolic inflammation. Costimulatory recep- in ATMs regulates MHC II and CD86 expression to control tors, such as CD40 and the B7 complex (CD80/CD86), are CD4+ T cell numbers in fat. induced in adipose tissue of obese humans and animal models [11–13]. However, the role of these costimulatory molecules in the development of adipose tissue inflammation and metabolic MATERIALS AND METHODS dysfunction remains cloudy. Some studies have identified a fi Mice and BMT studies bene cial role for costimulation in maintaining a Treg pool, both 2 2 CD40 / (B6.129P2-Cd40tm1Kik/J; C57BL/6Ncr background) mice and systemically and in adipose tissue, which is protective against fl congenic C57BL/6J mice (CD45.1 and CD45.2) were purchased from the obesity-induced in ammation and metabolic disease [5, 13]. In Jackson Laboratory (Bar Harbor, ME, USA). Lines were established and many other instances, loss of costimulation has been shown to maintained at the University of Michigan (Ann Arbor, Michigan, USA). All alter energy use and inflammation in animal models [14–19]. animal procedures complied with the Guide for the Care and Use of Laboratory CD40 is a surface glycoprotein, is expressed in hemato- Animals from the Institute of Laboratory Animal Research (Washington, DC, poietic and nonhematopoietic cells, and is activated by USA), and the University Committee on Use and Care of Animals at the University of Michigan approved all animal protocols. Beginning at 8 wk of CD40L/CD154 expressed on T cells and by soluble CD40L 2 2 age, male, WT C57BL/6J and CD40 / (KO) mice were fed an ND (4.5% fat; [20, 21]. CD40-CD40L signaling has an important role in PMI Nutrition International, St. Louis, MO, USA) or an HFD (60% fat; the regulation of APC function and the ability to stimulate Research Diets, New Brunswick, NJ, USA) to induce obesity. adaptive immunity [22]. In APCs, CD40 activation induces the BMTs were performed as described [32]. Male recipient WT CD45.1 mice expression of MHC II, CD80, and CD86 [23] and enhances (8 wk old) were exposed to a single dose of lethal irradiation (900 Rad) 4 h and chemokine production [24]. Relevant to obesity- before receiving 1 3 107 bone marrow cells (i.v.) from either WT (CD45.2) or fi research, serum levels of soluble CD40L are highly correlated KO (CD45.2) male donor mice. Reconstitution was con rmed 6 wk after transplantation by monitoring CD45.1 (recipient) vs. CD45.2 (donor) with obesity and metabolic syndrome [25, 26]. Rodent studies fl fi fl expression on blood leukocytes by ow cytometry. Diet studies were initiated indicate that CD40 de ciency prevents vascular in ammation 6 wk posttransfer (14 wk old), and BMT-WT and BMT-KO mice were fed fi and atherosclerosis [17, 27, 28]. Importantly, CD40 de ciency either ND or HFD for 20 wk. has been shown to block the proinflammatory M1 activation of macrophages [28], and CD40 has been identified as a marker of Isolation of SVCs for immunophenotyping and ATMs in obesity [11]. cell culture Several studies have examined the role of CD40 in obesity- Adipose tissue SVCs were isolated, as previously described [33]. For cell fl induced in ammation, yet it remains unclear whether CD40/ culture experiments, SVCs were isolated from epididymal fat pads of male CD40L signaling has protective or deleterious roles in modulat- WT mice fed HFD for 16 wk. SVCs were enriched for CD45+ leukocytes ing adipose tissue inflammation and metabolic homeostasis using mouse CD45 microbeads (Miltenyi Biotec, San Diego, CA, USA) 6 [16, 17, 29, 30]. CD40 is expressed on leukocytes and adipose andplatedat13 10 cells/ml in DMEM containing 10% heat-inactivated tissue stromal cells and adipocytes [12, 15, 31], which have FBS. After 3 h, nonadherent cells were removed, and fresh medium was added. Adherent cells were cultured overnight before being treated multiple roles in the regulation of adipose tissue metabolism. fi fl with recombinant mouse CD40 ligand (CD40L; R&D Systems, Minneapolis, CD40 de ciency in T cells potentiated adipose tissue in amma- MN, USA) for 6 h. tion during obesity, suggesting a protective function for CD40 signaling [29, 30]. By contrast, a chemical blockade of Flow cytometry analysis CD40-TRAF6 signaling, but not signaling through TRAF2/3/5, Blood leukocytes were prepared as described [34]. Single-cell suspensions protected mice from obesity-induced insulin resistance and were prepared from spleen and by pressing tissues through a hepatosteatosis [17, 19]. Obviously, variable results in these 40-mm cell strainer (BD Falcon; BD Biosciences, Franklin Lakes, NJ, USA). studies may be attributed to differences in the diet used to induce SVCs, blood leukocytes, splenocytes, and thymocytes were suspended in 7 obesity, to environmental conditions, to differences in gut PBS/0.5% BSA (10 cells/ml) and incubated in Fc block (rat anti-mouse microbiota, or to the genetic background strains. Nonetheless, a CD16/CD32; eBioscience, San Diego, CA, USA) for 15 min on ice. Cells were stained with the indicated for 30 min at 4°C in the consensus for the role of CD40 in obesity-induced adipose tissue fl fl dark. Antibodies used for ow cytometry are provided in Supplemental in ammation has not been reached. Table 1. Foxp3 staining kits (eBioscience) were used according to the Our group is interested in the nature and function of APCs in manufacturer’s instructions. Stained cells were washed twice in PBS, fixed in adipose tissue, and we have found that ATMs are the primary 0.1% paraformaldehyde, and analyzed on a FACSCanto II Flow Cytometer functional APC in fat that promote obesity-induced inflammation (BD Biosciences) using FlowJo software (version 9.4; Tree Star, Ashland, [3, 4]. Here, we performed an independent evaluation of the OR, USA). importance of CD40 in obesity-induced metainflammation to determine whether CD40/CD0L signaling contributed to Isolation of T cells, stimulation, and staining for ATM–T cell cross talk in obese adipose tissue. We observed that CD154 (CD40L) 2 2 Cd40 / (KO) mice had impaired induction of conventional SVCs were isolated from epididymal fat pads of male WT mice fed an HFD + + for 16 wk. SVCs were enriched for CD4 leukocytes using mouse CD4 CD4 T cells (Tconv) in adipose tissue with obesity, without any microbeads (Miltenyi Biotec) and plated at 1 3 106 cells/ml in RMPI 1640 significant changes in T or CD8+ T cells. This was associated reg supplemented with 10% FBS, 10 mM HEPES, 1 mM glutamine, 1 mM sodium with a decrease in the expression of MHC class II and CD86 pyruvate, and 50 mm 2-mercaptoethanol. CD4+ splenocytes were also isolated on ATMs in obese KO mice. Bone marrow chimeras generated and treated in parallel as controls. CD4+ T cells were stimulated with PMA 2/2 from Cd40 donors also had reduced Tconv numbers during (50 ng/ml) and ionomycin (500 ng/ml) for 2 h and were then treated with

1108 Journal of Leukocyte Biology Volume 99, June 2016 www.jleukbio.org Morris et al. CD40 regulates adipose tissue T cells

Figure 1. CD40/CD40L expression in visceral adipose tissue of mice. Male C57BL/6 mice were fed ND or HFD for 20 wk to induce obesity. (A) Confocal microscopy images demonstrating CD40 (green) and CD11c (red) expression on ATMs in eWAT from lean (ND) and obese (HFD) mice. Staining with rat isotype (IgG2ak) was used as a control. (B) Representative scatterplots demon- strating CD40 expression on adipose tissue leuko- cytes (CD45+) in eWAT from lean mice. (C) Surface expression of CD40 on ATMs (blue, CD45+CD11BhighF4/80High), CD11BMidF4/80Mid SVCs (green), and non-ATMs (red, CD45+ 2 2 CD11B F4/80 ) in eWAT from ND mice. Fluo- rescence minus one staining (FMO) (2CD40 mAb; shaded) is shown. (D) Histogram showing CD154 (CD40L) expression levels on unstimu- lated (2 PMA; black) and stimulated (+ PMA; red) CD4+ ATTs isolated from eWAT of HFD-fed mice. Splenocytes were isolated and treated in parallel as a positive control (lower histogram). (B–D) The percentage (%; means 6 SEM) of cells in each gate is indicated.

monensin (2 mM) for an additional 1 h. T cells were collected, fixed, and weight) was administered (i.p.), and blood glucose levels were measured at permeabilized before staining with CD4 and CD154 antibodies. 0, 15, 30, 45, 60, 90, and 120 min after injection. For ITTs, human insulin (1 unit/kg of body weight; Humulin R; Eli Lilly, Indianapolis, IN, USA) was Gene expression analysis injected i.p., and blood glucose levels (mg/dl) were measured 0, 15, 30, 45, and 60 min after injection. Plasma insulin levels were measured using an Quantitative RT-PCR in mice was performed as described [34]. Adipose ultrasensitive mouse insulin ELISA kit (Crystal Chem, Downers Gove, IL, tissue was flash frozen in liquid nitrogen and stored at 280°C until analysis. 2 USA). HOMA-IR values were calculated as fasting insulin (pM/L) 3 fasting Relative gene expression was determined using the 2 DDCT method after glucose (mM/L)/22.5. normalizing to Arbp expression. Sequences for PCR primers are provided online (Supplemental Table 2). Gene expression of human adipose tissue was Human adipose tissue samples assessed using TaqMan probes (Applied Biosystems, Foster City, CA, USA). Participants (n = 20) were recruited from the University of Michigan Bariatric Surgery Program (clinical characteristics are provided in Supplemental Metabolic tests Table 3). All participants in this cohort were female. Recruitment and study Body weights were measured weekly during experimental periods. Blood protocols were approved by the institutional review board at the University of glucose levels (mg/dl) were measured using a glucometer (One Touch Ultra; Michigan Medical School. Omental adipose tissue biopsies were collected Bayer, Whippany, NJ, USA). Mice were fasted for 6 h (10:00 AM–4:00 PM) at the time of surgery, flash frozen, and stored at 280°C before analysis. before GTTs or ITTs were performed. For GTTs, D-glucose (0.7 g/kg body Patients with a prior diagnosis of diabetes or taking one or more diabetic

www.jleukbio.org Volume 99, June 2016 Journal of Leukocyte Biology 1109 Figure 2. CD40L signaling induces the expression of APC genes in ATMs. SVCs were isolated from the eWAT of HFD mice and enriched for ATMs by positive selection (CD45+ microbeads) and plastic adherence. ATMs were cultured overnight and then treated with soluble recombinant mouse CD40L (10 mg/ml) for 6 h. (A–B) Relative expression of inflammatory and APC genes in the absence (CON) or presence of CD40L (n = 4 individual mice/treatment). Data are means 6 SEM.*P , 0.05, **P , 0.01, ***P , 0.001.

medications at the time of surgery were considered to be diabetic (n = 9). SVCs expressed detectable levels of CD40 (Fig. 1C); this compart- Patients were scored for presence or absence of physician-diagnosed ment contains B and T adipose tissue . A small metabolic syndrome criteria for hypertension and hyperlipidemia. percentage (;2%) of CD45+ CD11BMid F4/80Mid SVCs expressed CD40 (Fig. 1C); these were likely putative adipose tissue dendritic Statistical analysis cells, based on other reports [35]. Collectively, these data indicate Data are presented as means 6 SEM. GraphPad Prism software (version 5.01; that CD40 expressing ATMs are a major constituent of the stromal GraphPad Software, La Jolla, CA, USA) was used for statistical analysis. vascular compartment in eWAT from mice. Differences between groups were determined using unpaired, 2-tailed To support the model that CD40/CD40L signaling contributes Student’s t tests or 1-way ANOVA with Dunnett’s post hoc test. P , 0.05 was considered significant. The Pearson correlation coefficient was used to to interactions between ATMs and adipose tissue T cells, we + determine the relationship between genes expressed in human adipose tissue assessed expression of CD154/CD40L on CD4 ATT cells. CD154 samples. expression on CD4+ T cells is transient and is detectable only in activated T cells in many conditions [36–38]. We were unable to detect CD154 on freshly isolated CD4+ ATTs (data not shown); RESULTS however, when we stimulated isolated CD4+ ATTs and spleno- CD40/CD40L expression in adipose tissue of mice cytes from HFD-fed mice with PMA/ionomycin ex vivo, approximately one-half of the CD4+ ATTs expressed CD154 (Fig. We have previously shown that Cd40 is expressed in FAC-purified 1D). Taken together, these data suggest that CD40/CD40L ATMs and that obesity and inflammatory signals induce Cd40 signaling molecules may contribute to cross talk between ATMs expression in ATMs [3]. Given the diverse range of cell types and ATTs in adipose tissue. known to express Cd40 in adipose tissue, we used confocal microscopy and flow cytometry analysis to better delineate the expression of CD40 protein in situ. By confocal microscopy, CD40L signaling induces the expression of APC genes CD40 was detectable on ATMs in the visceral/eWAT from lean in ATMs mice fed an ND (Fig. 1A; upper panel). CD40 expression was In mice, alternative splicing of the pre-Cd40 mRNA generates 2 detected in both CD11c+ (M1) and CD11c (M2) ATMs in obese multiple CD40 isoforms, with and without intact intracellular fat after feeding HFD for 20 wk (Fig. 1A; middle panel). Low-level domains that convey signaling [34, 39]. Notably, these spliced CD40 immunoreactivity was observed on adipocytes by this variants are not easily distinguishable by the quantitative PCR method, but signals were significantly lower than on leukocytes, strategy that we used to examine Cd40 expression in previous suggesting that ATMs are the primary cell type expressing CD40 studies. Therefore, to determine whether CD40 expressed on protein in adipose tissue. ATMs is signaling competently, we treated cultured ATMs with We next examined CD40 expression on SVCs in eWAT from lean soluble recombinant mouse CD40L and evaluated the expression mice by flow cytometry. We found that CD40 is expressed almost of a number of CD40-regulated genes that are associated with exclusively by CD45+ SVCs(Fig.1B).ATMs(CD45+ CD11BHigh F4/ APC function. CD40L treatment induced robust increases in Il6, 80High)wereidentified as the predominant CD40+ leukocyte Il12p35, Il12p40, and Tnfa mRNA levels in ATMs from lean mice population in eWAT, and ;64% of ATMs expressed high levels of (Fig. 2A). The expression of H2-Ab1, the major MHC class II loci CD40 (Fig. 1C). Approximately 12% of CD45+ CD11BNeg F4/80Neg in C57BL/6J mice, as well as costimulatory molecules Cd80 and

1110 Journal of Leukocyte Biology Volume 99, June 2016 www.jleukbio.org Morris et al. CD40 regulates adipose tissue T cells

Figure 3. HFD-induced weight gain, but not visceral adiposity, is reduced in CD40-KO mice. (A) Body weight gain (g) in male WT and KO mice fed ND or HFD for 18 wk (n =8–11 mice/group). (B) Body weights in male WT and KO mice after 20 wk of diet exposure (n =8–11 mice/group). (C) eWAT, iWAT, and liver weight (g) in WT and KO mice fed ND and HFD for 20 wk (n =8–11 mice/group). (D) eWAT, iWAT, and liver weight expressed as a percentage of body weight. (E) Assessment of adipocyte area and size distribution in obese WT and KO mice. Data are means 6 SEM. *P , 0.05, **P , 0.01, ***P , 0.001. #P , 0.05 vs. ND-fed mice.

Cd86, was also induced by CD40L (Fig. 2B). These findings this difference in total body weight, HFD KO mice had larger indicate that CD40 activation may have a role in potentiating eWAT depots and smaller livers compared with WT mice APC function of ATMs. (Fig. 3C–D), suggesting improved efficiency of nutrient storage in visceral eWAT depots. By contrast, there were no significant CD40 is required from maximal accumulation of differences in subcutaneous/iWAT in KO and WT mice. + CD4 ATTs, but not ATMs, in response to Mean adipocyte size did not differ significantly between KO and diet-induced obesity WT mice (Fig. 3E). However, differences in adipocyte size To determine the role that CD40 has in obesity-induced adipose distribution were noted, with more small adipocytes in the WT 2 2 tissue inflammation, we fed WT and Cd40 / (KO) mice an mice and more moderate-sized adipocytes in the KO mice. HFD for 20 wk to induce obesity. Both WT and KO mice gained We next evaluated whether CD40 has a role in the regulation weight on the HFD, but KO mice gained less weight in of ATTs and ATMs in eWAT during HFD-induced obesity. For response to HFD compared with WT mice (weight gain: WT, 26.6 this, we used flow cytometry to identify and quantify ATT and 6 3.3g; KO, 23.3 6 2.5g; P = 0.03) (Fig. 3A–B). Metabolic ATM populations (see Supplemental Fig. 1 for gating strategies). phenotyping did not detect significant differences in energy Data are presented both as the percentage of SVCs and as the expenditure, energy consumption, or locomotor activity that total cell counts normalized to total fat pad weight to account for could explain the differences in body weights between the 2 the adipose tissue hypertrophy seen in HFD-fed mice. In lean genotypes on either ND or HFD (data not shown). Despite ND-fed mice, KO mice had an increase in the proportion of total

www.jleukbio.org Volume 99, June 2016 Journal of Leukocyte Biology 1111 Figure 4. CD40 is required from maximal accumulation of CD4+ ATTs in response to diet-induced obesity. WT and KO mice were fed ND and HFD, respectively, for 20 wk (n=5–8 mice/group). ATTs were identified and quantified in eWAT by flow cytometry (see Supplement Fig. 1 for gating strategy). (A) Quantitation of CD3+ ATTs. Data are expressed as the percentage of total SVCs and total cells/gram of adipose (eWAT) tissue. – + + + + fl 2 + + (B C) Quantitation of CD3 CD8 ATTs, and CD3 CD4 ATTs. (D) Representative ow plots demonstrating Tconv (Foxp3 ) and Treg (Foxp3 ) CD4 + fi + + 2 ATTs in eWAT from WT and KO mice fed an HFD. Percentage of CD4 ATTs in each gate is indicated. (E) Quanti cation of Tconv (CD3 CD4 Foxp3 ) + + + fi + + and Treg (CD3 CD4 Foxp3 ) ATTs. Ratio of Tconv:Treg ATTs was also determined. (F) Quanti cation of CD4 T cells and CD8 T cells in spleens from fi 6 , HFD-fed mice. G: Quanti cation of Tconv and Treg cells, and ratio of Tconv:Treg in spleens from HFD-fed mice. Data are means SEM.*P 0.05, **P , 0.01, ***P , 0.001. #P , 0.05 vs. ND-fed mice.

+ fi CD3 lymphocytes in eWAT (Fig. 4A). When normalized to a signi cant increase in the number of Tregs in eWAT in the lean adipose tissue weight, HFD induced a similar expansion of KO mice (Fig. 4E). HFD exposure induced an increase in the total lymphocytes in eWAT in both WT and KO mice. ND-fed number of Tconvs in eWAT of WT mice, whereas obese KO mice + fi mice had a similar proportion of CD8 lymphocytes in eWAT, but had signi cantly fewer Tconvs (Fig. 4E). In contrast, Treg content KO mice had more CD4+ lymphocytes (Fig. 4B–C). This in eWAT was comparable in both HFD-fed KO and WT mice difference was lost when normalized to fat mass. HFD exposure (Fig. 4E). In both diet conditions, the ratio of Tconvs:Tregs in induced similar accumulation of CD8+ lymphocytes in both eWAT was significantly decreased in KO mice (Fig. 4E). Again, genotypes. However, increases in CD4+ ATTs were seen in this effect was specific for adipose tissue because KO mice had

HFD-fed WT mice, but not in KO mice. When normalized to fat slightly more splenic Tconvs and fewer Tregs compared with WT mass, HFD-fed KO mice had a significant reduction in the mice (Fig. 4G). These data suggest that, in lean states, CD40 + number of CD4 ATTs compared with WT mice (Fig. 4C). These may serve to limit Treg numbers in adipose tissues. With HFD fi changes in T cells were speci c to eWAT because KO and WT exposure, the expansion of a subset of Tconvs in eWAT is dependent mice had similar numbers of blood (data not shown) and splenic on CD40, which may differ from other secondary lymphoid organs. CD4+ and CD8+ T cells in both diet conditions (Fig. 4F). We next assessed the influence of CD40 deficiency on ATM We next evaluated the distribution of CD4+ ATTs in content in eWAT. There were no differences in total ATM eWAT by assessing Foxp3 expression to differentiate Tconv content in eWAT from lean mice between genotypes (Fig. 5A). + + 2 + + + fi (CD3 CD4 Foxp3 )andTregs (CD3 CD4 Foxp3 ) (Fig. 4D). In With HFD, both WT and KO mice had signi cant increases in ND-fed mice, there were no difference in Tconv, but there was ATMs, but there was no difference in ATM numbers between

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of CD11c+ ATMs or the regulation of inflammatory genes in eWAT with HFD-induced obesity.

CD40 regulates MHC class II expression on ATMs during HFD-induced obesity MHC class II expression is induced on ATMs in eWAT during diet-induced obesity [3] and plays a pivotal role in regulating ATT numbers in mice [4]. Given our observations that CD40L stimulated H2-Ab1 and Cd86 expression in cultured ATMs (Fig. 2), we examined the expression of these molecules on ATMs in HFD-fed obese WT and KO mice. By gene expression analysis, CD40-deficiency was associated with a modest, but not statistically significant reduction in H2-Ab1 expression in total eWAT from lean and obese KO mice (Supplemental Fig. 2C). Flow cytometry analysis of ATMs from HFD-fed mice revealed that KO mice had a marked reduction in the frequency of MHC IIHigh CD86High ATMs and a corresponding increase in the frequency of MHC IILow CD86Low ATMs compared with WT mice (Fig. 6A–B). Compared with WT mice, the expression of MHC II and CD86 expression was also reduced on ATMs from KO mice (Fig. 6C–DandF–G). No differences in MHC II and CD86 expression were observed in CD11b+F4/80Low (puta- 2 2 tive adipose tissue dendritic cells) or CD11b F4/80 (non- ATMs) between obese WT and KO mice. Specificreductions in MHC II and CD86 expression were observed in both CD11c+ (recruited M1-like) and CD206+ (resident M2-like) ATMs (Fig. 6E and H). These findings indicate that CD40 is required for maximal MHC II and CD86 expression on ATMs during HFD-induced obesity. This reduction in MHC II and CD86 expression likely contributes to the impaired

accumulation of Tconv ATTs with diet-induced obesity in KO mice.

Hematopoietic CD40 promotes MHC class II Figure 5. CD40 deficiency does affect ATM infiltration into visceral fat expression on ATMs and accumulation of Tconv ATTs of mice during diet-induced obesity. WT and KO mice were fed ND during HFD-induced obesity – High and HFD for 20 wk (n=8 11 mice/group). ATMs (CD11B F4/ Given that CD40 is expressed on multiple cell types, we sought to High fi fl 80 ) were quanti ed in eWAT by ow cytometry (see Supplement evaluate the specific role of Cd40 in hematopoietic cells on Fig. 2 for gating strategy). (A) Quantification of ATMs. Data are adipose tissue inflammation. BMT experiments were performed expressed as the percentage of total SVCs and total cells/gram of adipose (eWAT) tissue. (B) Quantification of CD206+ (M2-like) ATMs. by reconstituting WT recipients with marrow from WT and KO + (C) Quantification of CD11c (M1-like) ATMs. Data are means 6 SEM. donors to generate chimeric mice with (BMT-WT) or without *P , 0.05. #P , 0.05 vs. ND-fed mice. (BMT-KO) CD40 expression on hematopoietic cells. Six weeks after transplantation, BMT-WT and BMT-KO mice were fed an ND or an HFD for 20 wk to induce obesity. BMT-WT and obese WT and KO mice. In both WT and KO mice, there was a BMT-KO mice had similar leukocyte chimerism in bone marrow, similar reduction in the proportion of resident CD206+ ATMs spleen, eWAT, and blood leukocyte compartments (as a and an increase in the number of CD11c+ ATMs in eWAT in percentage of their total leukocytes) after reconstitution (Sup- response to HFD (Fig. 4B–C). plemental Fig. 3). BMT-WT and BMT-KO mice did not differ in We assessed the expression levels of genes implicated in weight in both ND- and HFD-fed conditions (Fig. 7A). Obese adipose tissue inflammation (Ccl2, Tnfa, Il10, Nos2, Il12p35, BMT-KO and BMT-WT mice had comparable eWAT weight Il12p40, and Ifng). Irrespective of diet, there was no significant (Fig. 7B) and similar numbers of ATMs (data not shown). When difference in the expression levels of Ccl2, Tnfa, Il12p35, and we evaluated MHC II expression on ATMs from eWAT, we Il12p40 between WT and KO mice (Supplemental Fig. 2A–B). observedreducedMHCIIexpressiononATMsinbothND- Il10, Nos2, and Ifng expression levels were lower in eWAT from and HFD-fed BMT-KO mice compared with BMT-WT mice KO mice fed ND, but the expression of these genes was not (Fig.7C).ThisindicatesthatCD40deficiency leads to a different after HFD (Supplemental Fig. 3A–B). Collectively, cell-autonomous decrease in MHCII expression in ATMs. these data indicate that CD40 is required for the maximal When ATTs were examined, no significant quantitative + + + + + + expansion of a pool of CD4 Tconvs, but not for the recruitment differences in total CD3 , CD3 CD8 , or CD3 CD4 ATTs were

www.jleukbio.org Volume 99, June 2016 Journal of Leukocyte Biology 1113 Figure 6. CD40 regulates MHC class II expres- sion on ATMs during HFD-induced obesity. WT and KO mice were fed HFD for 20 wk. ATMs were analyzed by flow cytometry (see Supplement Fig. 2 for gating strategy). (A) Representative contour plots showing CD86 and MHC II (MHC I-A/I-E) expression on ATMs from WT and KO mice. The percentage (%) of cells in each gate is indicated. (B) Distribution (% of ATMs) of MHC IIHigh CD86High, MHC IIHigh CD86Low, MHC IILow CD86High, and MHC IILow CD86Low ATMs in eWAT from WT and KO mice fed HFD (n= 5–8 mice/group). (C) Representative histograms showing surface expression of MHC II (I-A/I-E) on ATMs from WT (black line) and KO (gray line) mice fed an HFD. Isotype staining is in- dicated in the shaded histogram. (D) MHC I-A/I- E expression (mean fluorescence intensity [MFI]) on ATMs, CD11BHighF4/80High ATMs, CD11B+F4/80Low SVCs, and Non-ATMs 2 2 (CD11B F4/80 ) after HFD exposure (n=5–8 mice/group). E. Surface expression (MFI) of MHC I-A/I-E on CD11c+ (M1-like) and CD206+ (M2-like) ATMs from WT and KO mice after HFD (n=5–8 mice/group). (F) Representative histograms showing surface expression of CD86 on ATMs from WT (black line) and KO (gray line) mice fed an HFD. Isotype staining is indicated in the shaded histogram. (G) CD86 expression 2 2 (MFI) on ATMs, CD11BHighF4/80High ATMs, CD11B+F4/80Low SVCs, and non-ATMs (CD11B F4/80 ) after HFD exposure (n=5–8 mice/group). (H) Surface expression (MFI) of CD86 on CD11c+ (M1-like) and CD206+ (M2-like) ATMs from WT and KO mice after HFD (n=5–8 mice/group). Data are means 6 SEM.*P , 0.05, **P , 0.01. observed between obese BMT-KO and BMT-WT mice (Fig. 7D). Coordinated regulation of CD40 and CD80/CD86 gene fi However, Tconv ATTs were signi cantly reduced in eWAT from expression in omental adipose tissue from obese obese BMT-KO mice compared with BMT-WT mice (Fig. 7E). By diabetic patients contrast, the number of Treg ATTs was comparable between To examine the relevance of CD40 in obese human adipose BMT-KO and BMT-WT mice fed either ND or HFD (Fig. 7E). tissue, we evaluated CD40, CD80, and CD86 gene expression in The overall ratio of Tconvs:Tregs was reduced in BMT-KO, HFD omental adipose tissue biopsies from a cohort of bariatric surgery mice compared with obese, BMT-WT mice. These observations patients (n = 20; clinical characteristics in Supplemental Table 3). with Tconv phenocopy observations in whole-body KO mice and There was no significant association with body-mass index and suggest that hematopoietic CD40 signaling is required for expression of any of these genes in this population (data not accumulation of Tconv ATTs with diet-induced obesity. shown). However, significant correlations were observed between CD40 and the expression of both CD80 and CD86 in the entire Whole-body, but not hematopoietic, CD40 deficiency cohort of obese patients (Fig. 9A–B). The patients were then improves insulin sensitivity during HFD-induced obesity subdivided based on the diagnosis of type 2 diabetes (non- We evaluated metabolic status of the mice generated in these diabetic, n = 11; diabetic n = 9). No significant differences in age experiments by assessing glucose tolerance and insulin sensitivity. or body-mass index were observed between the nondiabetic and Whole-body obese KO and WT mice had similar impairments diabetic groups. There were no differences in absolute expres- in glucose tolerance and insulin sensitivity as assessed by GTT sion of CD40, CD80, and CD86 between diabetic and nondiabetic and ITT (Fig. 8A–B). However, although HFD-fed KO and WT groups (data not shown). However, the expression of CD40 and mice had similar fasting glucose levels (Fig. 8C), obese KO mice CD86 was marginally correlated (r = 0.6553; P = 0.0554) in the had lower serum insulin levels and subsequently lower HOMA-IR diabetic group, and there was a significant positive correlation scores compared with obese WT controls (Fig. 8D). This is (r = 0.8212; P = 0.0066) between CD40 and CD80 expression in consistent with an increase of in vivo insulin sensitivity in KO omental fat from obese diabetic patients (Fig. 9C-D). By contrast, mice. However, when the bone marrow chimeras were analyzed, correlation between CD40 and CD80 or CD86 expression did not no significant differences in glucose tolerance, insulin tolerance, reach significance in the nondiabetic group. Thus, the associa- fasting glucose or insulin levels, or HOMA-IR were observed tion between CD40 and CD80/CD86 expression in omental fat BMT-KO and BMT-WT mice (Fig. 8E–H). These data suggest from this obese cohort is driven primarily by a strong coordinate that CD40 signaling in nonhematopoietic cells promotes whole- regulation of these genes in obese diabetic patients. These body insulin resistance. data demonstrate a coordinated regulation of costimulatory

1114 Journal of Leukocyte Biology Volume 99, June 2016 www.jleukbio.org Morris et al. CD40 regulates adipose tissue T cells

Figure 7. Hematopoietic CD40 promotes MHC class II expression on ATMs and accumulation of Tconv ATTs during HFD-induced obesity. Bone marrow cells from donor WT or KO mice were transplanted into lethally irradiated, male, WT recipients to generate mice with (BMT-WT) or without (BMT-KO) CD40 expression on hemato- poietic cells. Both groups were fed an ND or HFD for 20 wk. (A) Body weight gain in BMT-WT and BMT-KO mice fed an ND (n = 4 mice/group) or an HFD (n = 7 mice/group). (B) eWAT weight (g). (C) MHC I-A/I-E expression (mean fluores- cence intensity [MFI]) on ATMs from BMT-WT and BMT-KO mice fed an ND or an HFD for 20 wk. (D) Quantification of CD3+, CD3+CD8+, and CD3+CD4+ ATTs in eWAT. (E) Quantification of + + Tconv and Treg CD3 CD4 ATTs and the ratio of 6 Tconv:Treg ATTs in eWAT. Data are means SEM. *P , 0.05. #P , 0.05 vs. ND-fed mice.

2/2 genes in obese human adipose tissue, which may be a useful that Cd40 mice have decreased adipose tissue Tconv relative to biomarker for distinguishing metabolically healthy and unhealthy Tregs with obesity, which is mainly dependent on CD40 obese individuals. expression in hematopoietic cells. CD40 deficiency was also associated with reduced MHC II and CD86 expression on both M1-like (CD11c+) and M2-like (CD206+) ATMs in eWAT DISCUSSION 2 2 Cd40 / mice. Because ATMs are the primary APC in adipose The emerging concept that activation of the adaptive immune tissue [3, 4], the APC functionality of ATMs is at least partially system links obesity to metabolic disease has led our group and dependent on CD40 signaling to generate and maintain of others to examine the role of leukocyte cross talk in adipose Tconv in eWAT. Certainly, the reductions in Tconv content in 2 2 tissue inflammation [14, 40–43]. T cell costimulation is impor- obese Cd40 / mice parallels our recent observations that tant in APC–T cell communication, and these signals are often macrophage-specific deletion of MHCII in mice blunts the critical nodes in the regulation of both Treg and effector/ accumulation of Tconv in fat with obesity [4]. Notably, the memory Tconv activity and homeostasis [18, 44, 45]. Because the increase in Tregs in lean whole-body KO mice was not presence or absence of appropriate costimulatory signals likely recapitulated in BMT-KO mice, suggesting that hematopoietic influences the magnitude of adipose tissue inflammation in and nonhematopoietic CD40 may have divergent effects on obesity, many recent studies have examined the role of the regulation of Treg content in adipose tissue. Our data are in 2/2 costimulatory molecules in this process with somewhat mixed line with a report that shows an increase in Tregs in Cd40 results. This is likely due to the complexity of costimulatory mice [16, 47] but has not been the case in other studies [17, receptors and ligands that are expressed on the surface of both 30]. These findings are also in agreement with the observation fi leukocytes and nonleukocytes in adipose tissue. Additionally, that CD40L de cientmicehaveincreasedTregs and show soluble costimulatory molecules, such as CD40L and CD137L, are protection from insulin resistance with obesity [14]. Nonethe- increased in obesity and provide another level of costimulatory less, we observed that these differences are specific to adipose regulation [15, 25, 46]. tissue, which is in line with the unique nature of adipose tissue

Here, we examined the role of CD40 in the regulation of Tregs and their unique functional capacity compared with other fl adipose tissue in ammation in diet-induced obesity. We found induced Treg populations (e.g., spleen) [48, 49].

www.jleukbio.org Volume 99, June 2016 Journal of Leukocyte Biology 1115 Figure 8. Whole-body, but not hematopoietic, CD40 deficiency modestly improves insulin re- sistance during HFD-induced obesity. (A) Glucose excursion curve and area under the curve (AUC) analysis for GTT performed on WT and KO mice after 15 wk of HFD exposure (n = 8 WT mice; n = 11 KO mice). (B) Glucose excursion curve for ITT performed on WT and KO mice after 18 wk of HFD exposure (n = 8 WT mice; n =8KO mice). (C) Fasting (6 h) blood glucose and serum insulin levels in WT and KO mice after 19 wk on HFD (n = 8 WT mice; n = 8 KO mice). (D) HOMA-IR calculated for WT and KO male mice after 19 wk on HFD. (E) GTT and AUC analysis for BMT-WT and BMT-KO mice after 15 wk of HFD exposure (n = 7 mice/group). (F) ITT performed on BMT-WT and BMT-KO mice after 18 wk of HFD exposure (n = 7 mice/ group). (G) Fasting (6 h) blood glucose and serum insulin levels in BMT-WT and BMT-KO mice after 19 wk on HFD (n = 7 mice/group). (H) HOMA-IR calculated for BMT-WT and BMT- KO male mice after 19 wk on HFD. Data are means 6 SEM.*P , 0.05.

Changes in Tconv content have not been observed in other informative for understanding the leukocyte dynamics, so we + studies that have mainly focused on CD8 cells and Tregs. There is present both here. fi signi cant variation in how ATT content in adipose tissue is Despite a reduction in Tconvs, we observed only mild differ- reported in the literature, which makes it difficult to compare ences in metabolism based on glucose or insulin sensitivity in our results with other studies. Studies report ATT cells as total whole-body Cd40 deficient mice and no changes in BM chimeras 2 2 cells per adipose tissue fat pad, percentage of adipose tissue made from Cd40 / donors. Whole-body disruption of Cd40 leukocytes, or after normalizing to fat pad weight [16, 17, 30, in C57BL/6 mice offered modest protection from HFD-induced 47]. This remains a somewhat controversial issue, and there is weight gain and was associated with mild improvement in no clear, standardized measure of adipose tissue leukocyte insulin sensitivity, as determined by decreased circulating insulin 2 2 content. For example, we found that HFD-fed Cd40 / mice levels and improved HOMA-IR. This weight difference was 2 2 harbor more CD8+ ATTs as a percentage of SVCs, which is in not observed in BM chimeras generated with Cd40 / donors. line with other reports; however, when CD8+ ATT content was These observations differ from other studies that have shown 2 2 normalized to adipose tissue mass, there was no statistically both protection and aggravation of insulin resistance in Cd40 / significant difference between obese KO and WT mice. mice [16, 17, 30]. Different husbandry and diet conditions Given the massive expansion in adipose tissue weight with among laboratories may contribute to this variation, or it may HFD feeding and the increase in leukocytes within the SVCs, relate to studies demonstrating divergent effects of TRAF we feel that some degree of normalization is required. In our signaling pathways downstream of CD40 activation, with view, reporting ATT cells as a percentage of the all SVCs CD40–TRAF6 signaling promoting insulin resistance and CD40 and total ATTs per gram adipose tissue are the most signaling through TRAF2/3/5 having a protective effect on

1116 Journal of Leukocyte Biology Volume 99, June 2016 www.jleukbio.org Morris et al. CD40 regulates adipose tissue T cells

Figure 9. CD40 expression is positively correlated with expression of costimulatory genes (CD80, CD86) in omental adipose tissue of obese diabetic patients. RNA was isolated from omental adipose tissue biopsies from obese patients (n = 20) undergoing bariatric surgery. Expression of CD40, CD80, and CD86 was assessed by quantitative real- time PCR, and correlation analysis was performed. (A–B) Correlation analysis (Pearson) in the entire cohort (n = 20). (C–D) Correlation analysis of patients with the diagnosis of type 2 diabetes (diabetic; n = 9) and nondiabetic obesity (n = 11). Pearson correlation coefficients and P-value are shown for the diabetic group. Correlations within the nondiabetic group were not significant.

metabolism [17, 19]. Certainly, CD40 expression on non- Overall, our studies reveal a novel role for CD40 signaling hematopoietic cells is an important contributor. The lack of in hematopoietic cells. CD40 signaling promotes the significant metabolic changes in our obese KO mice parallels APC functionality of ATMs by controlling MHC II and the observation that ATM content, ATM phenotype, and the CD86 expression. We propose that this has an important expression of inflammatory were not different when role in translating obesogenic cues into an acquired compared with HFD-fed WT mice. Given that ATMs are potent immune response associated with the expansion of Tconvs in effectors of both innate and acquired signals in adipose tissue, adipose tissue. However, in this study, CD40-dependent fi it appears that the activation of ATMs in KO mice may be generation of Tconvs did not have a signi cant effect on the largely independent of CD40. development of insulin resistance, which is likely attributed to The coregulation of CD40 and costimulatory genes was a sustained activation profile for ATMs. This does not recapitulated in human visceral adipose tissue samples, where we exclude the possible use of inhibitors of the CD40/CD40L observed positive correlations between CD40 and CD80/CD86 pathway as a possible therapeutic strategy for metabolic expression in obese patients with type 2 diabetes. This disease but suggests additional pathways to interrogate in the observation agrees with gene expression profiling studies that process. have revealed associations between APC genes and obesity and metabolic disease in humans [50] and the ability of CD40 activation to induced CD80/86 expression [51]. Few AUTHORSHIP data exist on the expression of these genes in adipose tissue of diabetic patients, and cellular analyses have produced mixed D.L.M. conceived the study, designed and performed experi- results. CD80+ and CD86+ ATMs were shown to be negatively ments, analyzed data, and wrote the manuscript. K.E.O., T.A.M., associated with insulin resistance in human samples [13]. Others K.W.C., J.L.D., K.S., and R.W.O. performed experiments, have reported CD86 expression on ATMs to be an M1 macrophage acquired and analyzed data, and contributed to the discussion. marker that does not change significantly with obesity [52]. At the C.E.M., R.W.O., and C.N.L. contributed to the discussion. C.N.L. same time, CD86 is a member of a macrophage-enriched metabolic also wrote the manuscript, had full access to all the data in the network of genes shown to be increased in adipose tissue during study, and takes responsibility for the integrity of the data and metabolic disease [53–55]. the accuracy of the analysis.

www.jleukbio.org Volume 99, June 2016 Journal of Leukocyte Biology 1117 ACKNOWLEDGMENTS 12. Poggi, M., Jager, J., Paulmyer-Lacroix, O., Peiretti, F., Gremeaux, T., Verdier, M., Grino, M., Stepanian, A., Msika, S., Burcelin, R., de Prost, D., This work was supported by research grants from the U.S. National Tanti, J. F., Alessi, M. C. (2009) The inflammatory receptor CD40 is expressed on human adipocytes: contribution to crosstalk Institutes of Health (NIH) (Grants DK090262 [C.N.L], DK095050 between lymphocytes and adipocytes. Diabetologia 52, 1152–1163. [R.W.O], DK097449 [R.W.O], and DK093954 [C.E.M]), the 13. Zhong, J., Rao, X., Braunstein, Z., Taylor, A., Narula, V., Hazey, J., Mikami, D., Needleman, B., Rutsky, J., Sun, Q., Deiuliis, J. A., Satoskar, American Diabetes Association (Grant 7-12-CD-08 to C.N.L.), and A. R., Rajagopalan, S. (2014) T-cell costimulation protects obesity- the Indiana Health Clarian Values Research Fund (C.E.M.). D.L.M. induced adipose inflammation and insulin resistance. Diabetes 63, 1289–1302. was supported Grants DK091976 and DK100515 from NIH/ 14. Poggi, M., Engel, D., Christ, A., Beckers, L., Wijnands, E., Boon, L., National Institute of Diabetes and Digestive and Kidney Disease. Driessen, A., Cleutjens, J., Weber, C., Gerdes, N., Lutgens, E. (2011) fi fl K.S. was supported by an institutional K12 training grant from NIH CD40L de ciency ameliorates adipose tissue in ammation and metabolic manifestations of obesity in mice. Arterioscler. Thromb. Vasc. Biol. (HD028820) and a Pediatric Endocrine Society Research Fellow- 31, 2251–2260. ship Award. This worked used Core Services from the Michigan 15. Chatzigeorgiou, A., Phieler, J., Gebler, J., Bornstein, S. R., Chavakis, T. (2013) CD40L stimulates the crosstalk between adipocytes and Nutrition and Obesity Research Center (NIH Grant DK089503 to inflammatory cells. Horm. Metab. Res. 45, 741–747. the University of Michigan). We thank Drs. Mark Kaplan and 16. Guo, C. A., Kogan, S., Amano, S. U., Wang, M., Dagdeviren, S., Friedline, R. H., Aouadi, M., Kim, J. K., Czech, M. P. (2013) CD40 deficiency in Olufolakemi Awe, Indiana University School of Medicine, for mice exacerbates obesity-induced adipose tissue inflammation, hepatic providing technical assistance in detecting CD154 expression in steatosis, and insulin resistance. Am. J. Physiol. Endocrinol. Metab. 304, – restimulated CD4+ T cells, and Lynn Geletka and Dr. Gabriel E951 E963. 17. Chatzigeorgiou, A., Seijkens, T., Zarzycka, B., Engel, D., Poggi, M., van Martinez-Santibanez, University of Michigan, for helpful den Berg, S., van den Berg, S., Soehnlein, O., Winkels, H., Beckers, L., discussions. Lievens, D., Driessen, A., Kusters, P., Biessen, E., Garcia-Martin, R., Klotzsche-von Ameln, A., Gijbels, M., Noelle, R., Boon, L., Hackeng, T., Schulte, K.-M., Xu, A., Vriend, G., Nabuurs, S., Chung, K. J., Willems van Dijk, K., Rensen, P. C., Gerdes, N., de Winther, M., Block, N. L., Schally, A. V., Weber, C., Bornstein, S. R., Nicolaes, G., Chavakis, T., Lutgens, E. DISCLOSURES (2014) Blocking CD40-TRAF6 signaling is a therapeutic target in obesity- The authors declare no conflicts of interest. associated insulin resistance [published correction in Proc. Natl. Acad. Sci. U. S. A. (2014) 111, 464]. Proc. Natl. Acad. Sci. U. S. A. 111, 2686–2691. 18. Chatzigeorgiou, A., Chung, K. J., Garcia-Martin, R., Alexaki, V. I., Klotzsche-von Ameln, A., Phieler, J., Sprott, D., Kanczkowski, W., Tzanavari, T., Bdeir, M., Bergmann, S., Cartellieri, M., Bachmann, M., REFERENCES Nikolakopoulou, P., Androutsellis-Theotokis, A., Siegert, G., Bornstein, 1. Mathis, D. (2013) Immunological goings-on in visceral adipose tissue. Cell S. R., Muders, M. H., Boon, L., Karalis, K. P., Lutgens, E., Chavakis, T. Metab. 17, 851–859. (2014) Dual role of B7 costimulation in obesity-related nonalcoholic 2. Grant, R. W., Dixit, V. D. 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